Despite recent advances in both targeted therapy and immunotherapy, the overall survival for metastatic melanoma remains low. This renewal application is to continue our productive efforts to develop novel tubulin inhibitors and their targeted deliveries for more effective treatment of metastatic melanoma. In the first funding period, we identified several potent scaffolds and developed two nanoparticle formulations that significantly improved in vivo efficacy against melanoma metastasis. Our objectives in this renewal are to perform focused lead optimization using integrated medicinal chemistry, structural biology and molecular biology approaches, and to optimize nanoparticle formulations for high drug loading and nanoparticle kinetic stability. Aim 1: Perform focused lead optimization using the two best scaffolds and iteratively optimize their activities. Aim 1.1. Using the comprehensive SAR we have accumulated, we will perform focused lead optimization with the two best scaffolds to further improve their interactions with the colchicine binding site. Aim 1.2. We will screen new analogs using a panel of melanoma cell lines, including multidrug resistant phenotypes and melanoma colony formation. Melanocytes will serve as a model of normal cells to assess the selectivity. We will select up to the 20 best analogs for Aim 2. Aim 2: Confirm the modes of action, determine molecular interactions with tubulin, and assess drug- like properties. Aim 2.1. We will determine their ability to inhibit tubulin polymerization, quantitatively assess their binding to the colchicine site in tubulin, investigate their effects on apoptosis, and evaluate their anti- angiogenesis potential. Aim 2.2. We will determine molecular interactions between the best analogs and tubulin by solving the X-ray crystal structures of tubulin in complex with a diverse set of potent analogs. These results will feed back to Aim 1 to guide more efficient structural optimization. Aim 2.3. We will assess drug-like properties and off-target effects for the selected new analogs to select up to 6 of the best analogs for Aim 3. Aim 3: Optimize nanoparticle formulations, determine in vivo efficacy against melanoma tumor growth and metastasis using both free drugs and their optimal nanoparticle formulations. Aim 3.1. We will use hydrolyzable bonds to conjugate new analogs to polymer backbones before forming nanoparticles for higher drug loading capacity and improved kinetic stability of nanoparticles. We will incorporate melanoma tumor- specific peptides to the drug loaded nanoparticles for active targeting. Aim 3.2 We will evaluate in vivo efficacy against melanoma growth, metastasis, tumor angiogenesis, and potential toxicities. We will compare the efficacy of free drugs and their optimal nanoparticle formulations, using Abraxane (nanoparticle formulated paclitaxel) as the reference control. Aim 3.3. For the three most efficacious tubulin inhibitors an their nanoparticle formulations, we will determine their bio-distribution and pharmacokinetic profiles in mice after systemic drug administration.